Hepatitis C is a viral infection of the liver which has also been referred to as “non A, non B hepatitis” (NANBH) until identification of the causative agent. Hepatitis C virus is one of the viruses (A, B, C, D and E), which together account for the majority of cases of viral hepatitis. Hepatitis C virus was first identified in 1989 and defined as a common cause of liver disease with an estimated 170-million infected people worldwide. Hepatitis C virus (HCV) infection affects the liver, which causes hepatitis, i. e., an inflammation of the liver. 75 to 85% of persons infected with HCV progress to chronic infection, approximately 20% of these cases develop complications of chronic hepatitis C, including cirrhosis of the liver or hepatocellular carcinoma after 20 years of infection. The current recommended treatment for HCV infections includes a combination of interferon and ribavirin drugs, with either boceprevir or telaprevir added in some cases. Overall, 50-80% of people treated are cured. Those who develop cirrhosis or liver cancer may require a liver transplant. Hepatitis C is the leading cause of liver transplantation, though the virus usually recurs after transplantation. No vaccine against hepatitis C is available.
HCV is a (+) sense single-stranded enveloped RNA virus in the Hepacivirus genus within the Flaviviridae family. The viral genome is approximately 10 kilobases (kb) in length and encodes a 3011 amino acid polyprotein precursor. The HCV genome has a large single open reading frame (ORF) coding for a unique polyprotein, said polyprotein being co- and post-translational processed by cellular and viral proteases into three structural proteins, i. e., core, E1 (envelope) and E2 (envelope) and at least seven non-structural proteins; p7 (ion channel), NS2 (protease), NS3 (serin-protease/helicase), NS4A (cofactor), NS4B (replication complex factor), NS5A (interferon resistant protein) and NS5B (RNA dependent RNA polimerase).
HCV shows a high genetic variability. The reason for this great variation is a high mutation rate and high level of viral replication through an error-prone RNA polymerase without proofreading capacity. Analysis of extensive sets of sequences from HCV isolates throughout the world has revealed the existence of six major genetic groups or genotypes, and a large number of subtypes (also named subgenotypes) within the six main genotypes. Genotypes are numbered from 1 to 6 and subtypes designated as a, b, c, etc. (i.e.: 1a, 1 b, 2a, 2b, etc.) in both cases in order of discovery. Overall sequence divergence between genotypes ranges from 31 to 34% and from 20 to 23% between subtypes An extent discussion of the adopted basis for the classification of genotypes and subtypes, as well as the standard reference methods for genotyping, can be seen in the document of Simmonds et al., “Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes”, Hepatology—2005, vol. 42, pp. 963-973.
Determining the correct infection in terms of the infecting genotype (or even subtype) is fundamental in order to assign a correct treatment. Indeed, patients infected with genotypes 1 and 4 do not respond as efficiently to the standard therapy as the other genotypes, thus implying a longer therapy (48 weeks vs. 24 weeks). Or for example, genotypes 2 and 3 have great chances of response to the treatment with alfa-interferon than genotype 1. Moreover, each genotype has a different progression or development into the host. So that, genotype 1b has an accelerated development to cirrhosis than other genotypes. The genotype of an infected subject does not change during the infection history unless an additional infection takes place with another genotype or subtype.
There are several commercial kits being used for genotyping infected HCV patients, even kits that identify the subtype of a genotype. They differ in the fragment of the virus genome that is analyzed, as well as in the molecular biology tools employed in the determination of the HCV variant.
One of the methods for HCV genotyping is the Inno-Lipa HCV reverse hybridization assay (Innogenetics). In this assay, the 5′-UTR region of the virus is analyzed by reverse hybridization. Thus, non-coding 5′ segments are amplified and further hybridized with reference probes that allow the genotype determination. A more robust version of Inno-Lipa is the LIPA 2.0, in which an additional region of the virus is amplified and hybridized, namely the coding core region.
Another option is the TruGene HCV 5′NC genotyping assay (Bayer). By this technique a non-coding segment of the 5′ region is semi-automatically sequenced. 5′-UTR regions are amplified, sequenced and aligned with a reference sequence panel.
Also the Abbott method known as HCV genotyping ASR assay can be an option. In this case, a quantitative PCR of the 5′-UTR and NS5b regions is performed. The method proposes specific NS5b primers and probes for the detection of 1a and 1b sub-genotypes, and other 5′-UTR specific primers and probes for the genotypes and sub-genotypes 2a, 2b, 3, 4, 5 and 6.
All these methodologies appear disclosed in the document of Chevaliez S. et. al. “Hepatitis C virus (HCV) genotype 1 subtype identification in new HCV drug development and future clinical practice”, PLoS ONE, 2009, vol. 4, pp. 1-9.
The methodologies are usually compared with the reference method, which is the Sanger sequencing of the whole virus, or of at least the NS5B region or 5′UTR-core/E1 region (Simmods et al. supra). Although the sequencing is considered the reference method, it implies the disadvantages of miss-readings due to the fact that the resulting sequence corresponds to an average of the sequences in an isolated sample. Thus, co-infections (infections with more than one HCV subtype at the same time) cannot be detected. In addition, a cloning and sequencing method to detect co-infections implies long analytical times and expensive costs, thus making it no practical in commercial kits.
Although all the existing kits based on semi-automatically sequencing, quantitative PCR, or reverse hybridization, have a high success rate in the HCV determination, there are also some limitation making them no infallible and leading to some erroneous determinations.
In effect, there are some subtypes difficulty distinguishable between each other, for examples subtypes 1a and 1 b, and there exist some false positives and false negatives. In addition, the above-mentioned kits do not allow the determination of multiple infections (co-infections) in case the subject is infected with an accompanying additional genotype or subtype in a low load or proportion.
But, there exists also an additional level of complexity in the infections with HCV that makes much more difficult the correct determination of the genotype or sub-genotype (subtype), in particular the possibility of an infection with a recombinant virus. The existence of recombinant variants of HCV is due to the fact that in patients co-infected with more than one genotypes or subtypes, recombination takes place between all these variants leading to an “hybrid” genotype or subtype, making difficult the determination of the real infecting variants. As above indicated, the determination of the real variant is of special importance in order to adapt or better prescribe a medical treatment.
It is, hence, of great interest to further develop improved genotypic assays for detecting the specific variants in HCV infected patients in order to better diagnose the subjects, and in order to detect resistance-associated mutations of the virus with higher sensitivity, with the aim of further predicting the responses to HCV prescribed treatments.